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- PDB-9cra: CryoEM Structure of the C-terminally truncated form of human NAD ... -

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Basic information

Entry
Database: PDB / ID: 9cra
TitleCryoEM Structure of the C-terminally truncated form of human NAD Kinase bound to NAD
ComponentsNAD kinase
KeywordsSIGNALING PROTEIN / catalyzes the reaction of ATP + NAD+ = ADP + H+ + NADP+
Function / homology
Function and homology information


NAD+ kinase / NAD+ kinase activity / NADP+ biosynthetic process / Nicotinate metabolism / NAD+ metabolic process / phosphorylation / positive regulation of insulin secretion involved in cellular response to glucose stimulus / ATP metabolic process / ATP binding / metal ion binding / cytosol
Similarity search - Function
ATP-NAD kinase C-terminal domain / NAD kinase / ATP-NAD kinase, PpnK-type, C-terminal / ATP-NAD kinase N-terminal domain / NAD kinase/diacylglycerol kinase-like domain superfamily / Inorganic polyphosphate/ATP-NAD kinase, N-terminal
Similarity search - Domain/homology
NICOTINAMIDE-ADENINE-DINUCLEOTIDE / NAD kinase
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.34 Å
AuthorsLi, Y. / Chen, Z. / Mary, C. / Labesse, G. / Hoxhaj, G.
Funding support United States, France, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)R01GM143236 United States
Welch FoundationI-2067-20210327 United States
Welch FoundationI-2067-20240404 United States
Agence Nationale de la Recherche (ANR)ANR-20-CE18-0025 France
Fondation pour la Recherche Medicale (FRM)EQU202303016265 France
CitationJournal: Sci Adv / Year: 2025
Title: Cryo-EM structure and regulation of human NAD kinase.
Authors: Prakash P Praharaj / Yang Li / Charline Mary / Mona H Soflaee / Kevin Ryu / Dohun Kim / Diem H Tran / Trishna Dey / Harrison J Tom / Halie Rion / Muriel Gelin / Andrew Lemoff / Lauren G ...Authors: Prakash P Praharaj / Yang Li / Charline Mary / Mona H Soflaee / Kevin Ryu / Dohun Kim / Diem H Tran / Trishna Dey / Harrison J Tom / Halie Rion / Muriel Gelin / Andrew Lemoff / Lauren G Zacharias / João S Patricio / Thomas P Mathews / Zhe Chen / Corinne Lionne / Gerta Hoxhaj / Gilles Labesse /
Abstract: Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is a crucial reducing cofactor for reductive biosynthesis and protection from oxidative stress. To fulfill their heightened anabolic and ...Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is a crucial reducing cofactor for reductive biosynthesis and protection from oxidative stress. To fulfill their heightened anabolic and reductive power demands, cancer cells must boost their NADPH production. Progrowth and mitogenic protein kinases promote the activity of cytosolic NAD kinase (NADK), which produces NADP, a limiting NADPH precursor. However, the molecular architecture and mechanistic regulation of human NADK remain undescribed. Here, we report the cryo-electron microscopy structure of human NADK, both in its apo-form and in complex with its substrate NAD (nicotinamide adenine dinucleotide), revealing a tetrameric organization with distinct structural features. We discover that the amino (N)- and carboxyl (C)-terminal tails of NADK have opposing effects on its enzymatic activity and cellular NADP(H) levels. Specifically, the C-terminal region is critical for NADK activity, whereas the N-terminal region exhibits an inhibitory role. This study highlights molecular insights into the regulation of a vital enzyme governing NADP(H) production.
History
DepositionJul 21, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 8, 2025Provider: repository / Type: Initial release
Revision 1.1Feb 5, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Feb 12, 2025Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: NAD kinase
B: NAD kinase
C: NAD kinase
D: NAD kinase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)166,9338
Polymers164,2804
Non-polymers2,6544
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
NAD kinase / Poly(P)/ATP NAD kinase


Mass: 41069.910 Da / Num. of mol.: 4 / Fragment: C-terminal residues 91-437
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NADK / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: O95544, NAD+ kinase
#2: Chemical
ChemComp-NAD / NICOTINAMIDE-ADENINE-DINUCLEOTIDE


Mass: 663.425 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C21H27N7O14P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: NAD*YM
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: human NAD Kinase / Type: COMPLEX
Details: Tetramer of c-terminally truncated form of human NAD Kinase bound to NAD
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 49 kDa/nm / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Buffer solutionpH: 7.8 / Details: 50 mM Tris (pH 7.8), 50 mM NaCl, and 2 mM DTT.
Buffer component
IDConc.NameBuffer-ID
150 mMTRIS1
250 mMNaCl1
32 mMDTT1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Purified by FPLC. Monodispersed.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 9105
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2SerialEM4image acquisition
4cryoSPARCCTF correction
7UCSF ChimeraXmodel fitting
9PHENIXmodel refinement
10Cootmodel refinement
11cryoSPARCinitial Euler assignment
12cryoSPARCfinal Euler assignment
13cryoSPARCclassification
14cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1716164 / Details: template based picking using a low resolution map
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.34 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 251813 / Symmetry type: POINT
Atomic model buildingSpace: REAL

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